1. Field of the Invention
The present invention relates to novel nucleic acid sequences which encode an operon involved in L-arabinose utilization. The operon includes a promoter which is both inducible and repressible and can be used to promote expression of genes in prokaryotics.
2. Description of the Related Art
Bacillus subtilis, an endospore-forming Gram positive bacteria, is able to grow on L-arabinose as the sole carbon source. L-arabinose residues are found widely distributed among many heteropolysaccharides of different plant tissues, such as arabinans, arabinogalactans, xylans, and arabinoxylans. Bacillus species in its natural reservoir, the soil, participate in the early stages of plant material decomposition, and B. subtilis secretes three enzymes, and endo-arabanase and two arabinosidases, capable of releasing arabinosyl oligomers and L-arabinose from plant cell walls [Kaji and Seheki, Biochim. Biophys. Acta., 410:354-360 (1975); Weinstein and Albersheim, Plant Physiol., 63:425-432 (1979)]. The pathway of L-arabinose utilization in B. subtilis was described by Lepesant and Dedonder [C R Acad. Sci., Ser.D:2683-2686 (1967a)]. After entering the cell, L-arabinose is sequentially converted to L-ribulose, L-ribulose-5-phosphate, and D-xylulose-5-phosphate by the action of L-arabinose isomerase, L-ribulokinase and L-ribulose 5-phosphate 4-epimerase, respectively. D-xylulose-5-phosphate is further catabolized through the pentose-phosphate pathway. Mutants unable to use L-arabinose as sole carbon source, deficient in one of the three enzymes involved in L-arabinose catabolism, were characterized as well as constitutive mutants for all the three enzymes [Lepesant and Dedonder, 1967a, supra; Lepesant and Dedonder, C R Acad. Sci., Ser.D:2832-2835 (1967b)]. The synthesis of these enzymes was shown to be inducible by L-arabinose and the isomerase activity subjected to catabolite repression by glucose and glycerol [Lepesant and Dedonder, 1967a, supra].
A collection of Ara.sup.- B. subtilis mutants was isolated, biochemically characterized and the three metabolic genes, araA, araB and araD coding for L-arabinose isomerase, L-ribulokinase and L-ribulose 5-phosphate 4-epimerase, respectively, were identified and mapped between aroG and leuA, at about 256.degree. on the B. subtilis genetic map [Paveia and Archer, Broteria Genetica, Lisboa, XIII(LXXX):149-159 (1992a); Paveia and Archer, Broteria Genetica, Lisboa, XIII(LXXX):161-167 (1992b)]. Two additional classes of mutations affecting L-arabinose utilization were identified; one included mutations conferring and Ara.sup.- phenotype to strains bearing the araA, araB and araD wild types alleles [Paveia and Archer, 1992a, supra; Paveia and Archer, 1992b, supra] and another comprised mutants showing constitutive expression of the three genes [Sa-Nogueira et al., J. Bacteriol, 170:2855-2857 (1988)]. These mutations were mapped between the cysB and hisA markers, at about 294.degree. on the B.subtilis genetic map, and define another ara locus named araC. Expression of L-arabinose isomerase is severely repressed during growth in media containing L-arabinose plus glucose. Since L-arabinose isomerase expression is still regulated by catabolite repression in strains which contain constitutive mutations, araC.sup.C, L-arabinose transport does not play a major role in catabolite repression of expression of the metabolic enzymes [Sa-Nogueira et al., 1988, supra]. The genes araA, araB and araD, have been cloned and by complementation experiments its products were shown to be functionally homologous to their Escherichia coli counterparts. Transformation experiments involving defined restriction fragments from the cloned genes showed that they are adjacent and probably constitute an operon with the order A-B-D [Sa-Nogueira and Lencastre, J. Bacteriol., 171:4088-4091 (1989)], unlike the B-A-D order found in the E. coli operon [Englesberg et al., Proc. Natl. Acad. Sci. USA, 80:6790-6794 (1969)].
Expression of cloned genes introduced into bacteria has been, and is still, a mechanism for producing large amounts of a protein of interest for diagnostic and therapeutic purposes. In order to efficiently produce proteins in a prokaryotic host, a strong, regulated promoter is an essential element of the expression system.
Prokaryotic promoters used in the past include the bacteriophage lambda p.sub.L promoter, which is regulated by a temperature-sensitive repressor which represses transcription from that promoter at low temperatures. The p.sub.L promoter is used in an E. coli strain which contains a defective lambda prophage which encodes the repressor. This system is particularly suited for the expression of proteins which are toxic to E. coli. However, although the system is repressible, it does not provide a mechanism for inducibility.
Another prokaryotic promoter is the trp-lac promoter or tac promoter, which has been used to produce high levels of proteins in E. coli. This promoter is induced in the presence of isopropylthio-.beta.-D-galactoside (IPTG). However, in order to subject the promoter to repression it must be used in an E. coli strain which produces lac repressor protein.
The bacteriophage T7 promoter can be used to express proteins in bacteria which are not normally efficiently transcribed by E. coli RNA polymerase. However, this system requires the use of an exogenous T7 RNA polymerase, and may require the use of specialized host cells, or supplemental infection with a bacteriophage in order to maintain low expression levels of proteins which are toxic to E. coli.
Therefore, in view of the aforementioned deficiencies attendant with prior art methods of controlling gene expression in prokaryotic hosts, it is apparent that there exists a need in the art for a promoter system for the expression of exogenous DNA in prokaryotes, which is highly regulable, i.e., is both inducible and repressible.